Localized measurement of kinase activation in oocytes of Xenopus laevis

We have combined a rapid cytoplasmic sampling technique with capillary elec trophoresis to measure the activation of protein kinase C (PKC) in a small region (approximately 60 mu m) of a Xenopus oocyte, The phosphorylation of a fluorescent PKC substrate was measured following addition of a pharmacolo gical or physiological stimulus to an oocyte. When substrates for cdc2 kina se (cdc2K), PKC, and protein kinase A (PKA) were comicroinjected into an oo cyte, all three substrates could be identified on the electropherogram afte r cytoplasmic sampling. With this new method, it should be possible to meas ure simultaneously the activation of multiple different kinases in a single cell, enabling the quantitative dissection of signal transduction pathways .